GnRH receptor activation competes at a low level with growth signaling in stably transfected human breast cell lines

Abstract

BackgroundGonadotrophin releasing hormone (GnRH) analogs lower estrogen levels in pre-menopausal breast cancer patients. GnRH receptor (GnRH-R) activation also directly inhibits the growth of certain cells. The applicability of GnRH anti-proliferation to breast cancer was therefore analyzed.MethodsGnRH-R expression in 298 primary breast cancer samples was measured by quantitative immunofluorescence. Levels of functional GnRH-R in breast-derived cell lines were assessed using 125I-ligand binding and stimulation of 3H-inositol phosphate production. Elevated levels of GnRH-R were stably expressed in cells by transfection. Effects of receptor activation on in vitro cell growth were investigated in comparison with IGF-I and EGF receptor inhibition, and correlated with intracellular signaling using western blotting.ResultsGnRH-R immunoscoring was highest in hormone receptor (triple) negative and grade 3 breast tumors. However prior to transfection, functional endogenous GnRH-R were undetectable in four commonly studied breast cancer cell lines (MCF-7, ZR-75-1, T47D and MDA-MB-231). After transfection with GnRH-R, high levels of cell surface GnRH-R were detected in SVCT and MDA-MB-231 clones while low-moderate levels of GnRH-R occurred in MCF-7 clones and ZR-75-1 clones. MCF-7 sub-clones with high levels of GnRH-R were isolated following hygromycin phosphotransferase transfection. High level cell surface GnRH-R enabled induction of high levels of 3H-inositol phosphate and modest growth-inhibition in SVCT cells. In contrast, growth of MCF-7, ZR-75-1 or MDA-MB-231 clones was unaffected by GnRH-R activation. Cell growth was inhibited by IGF-I or EGF receptor inhibitors. IGF-I receptor inhibitor lowered levels of p-ERK1/2 in MCF-7 clones. Washout of IGF-I receptor inhibitor resulted in transient hyper-elevation of p-ERK1/2, but co-addition of GnRH-R agonist did not alter the dynamics of ERK1/2 re-phosphorylation.ConclusionsBreast cancers exhibit a range of GnRH-R immunostaining, with higher levels of expression found in triple-negative and grade 3 cancers. However, functional cell surface receptors are rare in cultured cells. Intense GnRH-R signaling in transfected breast cancer cells did not markedly inhibit growth, in contrast to transfected HEK 293 cells indicating the importance of intracellular context. GnRH-R signaling could not counteract IGF-I receptor-tyrosine kinase addiction in MCF-7 cells. These results suggest that combinatorial strategies with growth factor inhibitors will be needed to enhance GnRH anti-proliferative effects in breast cancer

Gonadotropin-releasing hormone receptor activates GTPase RhoA and inhibits cell invasion in the breast cancer cell line MDA-MB-231

“…In fact, the increased expression levels and IP response to GnRHa detected in GnRHR-transfected MDA cells, emulated those previously detected in breast cancer cells exhibiting high GnRHR expression levels [52]. Here we confirm that to detect relevant effects of GnRH on breast cancer cells function, it is necessary to substantially increase cell surface plasma membrane receptor levels, which is an important issue given that the number of GnRHRs is highly variable in malignant breast tumors tissue [51]. In this scenario, measurement of GnRHR density in malignant breast tissue may be useful as a surrogate marker to predict the tumor responsiveness to GnRHa administration.…”

“…To this end, we overexpressed the GnRHR in MDA cells and analyzed the effects of its cognate ligand on the pathways leading to actin cytoskeleton activation and cell adhesion. MDA cells transfected with the GnRHR (WT and DesK191) cDNAs, specifically bound 125 I]-Buserelin and produced higher levels of the second messenger IP in response to the GnRH analog than untransfected cells, overcoming the problem related to the low naturally expressed GnRHR levels in breast cancer cell lines [50,51]. In fact, the increased expression levels and IP response to GnRHa detected in GnRHR-transfected MDA cells, emulated those previously detected in breast cancer cells exhibiting high GnRHR expression levels [52].…”

Expression of a urokinase‐type plasminogen activator during tumor growth leads to angiogenesis via galanin activation in tumor‐bearing mice

“…However, lack of prohormone convertase and/or constitutive release was found to cause the release of these neuropeptides in a precursor form . In addition, there are many cases where these ectopic hormone‐producing tumors express their hormone‐specific receptors . However, precursor peptides usually show a low binding potential to their own specific receptors.…”

Inferences From Dysregulated Long Non-Coding RNA-Mediated Competing Endogenous RNAs in Various Chemotherapy Drugs and Evaluation of Drug Response in Breast Cancer

“…A large number of studies have reported that BRCA is well known for being strongly in uenced by female steroids [28][29][30]. Inositol phosphate is also thought to play an essential role in BRCA [31]. In addition, we explored the relationships between drug-resistant and -sensitive dysregulated ceRNAs and cancer hallmarks.…”